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stange cloning results - pBluescript seeming to double up (Jan/18/2006 )

I'm cloning and getting no where.
sticky end ligation, using Eco RI and Bam HI cut pBluescript (gel purified). 500 bp product (PCRed from genomic DNA, cut with EcoRI and BamHI, also purified). Ligated overnight, 3:1 ratio, all proper controls, ethanol precipitated ligation, electrotransformed into JM109s, recovery in SOC for 30 min. getting colonies.

it looks like the ligations have worked, ~10 fold more colonies on my plates compared to the controls (no T4, and no insert). Double digesting with HindIII and NotI to see if there is insert.
now here's the rub: cut pBluescript should run about 3kb. my vector is running at 6kb. my insert should be 500bp. on the gel, it's running at 1kb.
but the vectors that haven't accepted the insert, is running at 3kb.

it seems that everything has doubled up. can someone explain why this is happening, and what can be done about this?
or could there be a problem with the ladder?




I am assuming you are screening minis with hindIII and NotI?

If you have any of the vector/insert from before ligation or maybe the no ligation control you could run that on the gel with your digested minis to ensure that the sizes are as expected and your marker is good...

Do you get the "6kb" and "1kb" bands from the same miniprep? Are they from digested preps? If so, how does the uncut plasmid compare to uncut pbluescript?

I don't know about the pBluescript RE map, or your insert, but I think you shouldn't see the 6kb band after digestion b/c even if it is doubled up in the plasmid, you would digest them all to the original 3kb and 500bp pieces and just have 2X as much... If you post the location of RE sites you are using I will play around on paper with the possible combinations....


i've also cut the mini's with EcoRI and BamHI, and have exactly the same result (6kb band and a 1kb band).

i'm going to run the mini's (uncut) with pBluescript uncut, just to see what's going on.
i've also set up another ligation, and am going to run that out on a gel, to see where the problem lies.

at first i thought it was a contamination, but this is all new stuff, and the same thing keeps on happening (for 3 weeks now).

i've enclosed the map of the MCS. i can't see how i'm getting the results i am.
Attached Image

maybe the ladders off. i don't know. maybe one of the enzymes isn't cutting (before the ligation).. really clueless on this one.

i know the pbluescript it not contaminated, and is cut (maybe not with both?). i know the insert is not contaminated (but maybe uncut? unlikely .... i've followed the protocols, and have cut with these two enzymes before, and have had successful ligations from it.)
using a fresh T4 enzyme, and buffer.
water is clean.
possibly going insane.

Just finished running the gel of the uncut weird ligated plasmid with uncut pBluescript. My weird uncut ligated plasmid runs at ~10kb, and the uncut pBluescript at ~1.5 kb.
running a gel of the ligation tomorrow morning. dry.gif


Sorry I couldn't get back to you sooner... This is a puzzle, can't figure how unless multiple tested enzymes stopped working at the same time??? Any of the concatamers I can come up with should digest back to the original 3kb and 500bp pieces, however the fact you have 10kb uncut does seem to indicate some kind of concatamer though!?!?

The thing that really stumps is that both NotI HindIII and Bam EcoR give the same abarrent pattern, if one enzyme cut and the other didn't in the initial prep then the concatomers should be broken up into original pieces at least by cutting with the bam/ecor combination in the minis...

if the PCR product didn't digest then you would be looking at a T/A tailed or blunt end product and it shouldn't ligate at all to the sticky end vector... I would get into something like template amount from gDNA, but if the template was interfering I think you would have a lot of colonies with different insert sizes, and it doesn't help with the 6kb vector problem....

I figure you have thought of all of this, just helps me think to write it out... If you haven't solved yet then I think I would try single digestion, I may even switch to an enzyme that cuts in my insert but not in the vector for this, and then compare linear sizes...

At this point I am not so sure I don't question your marker too!!! Do you have another available in lab to run simultaneously to check that, or maybe linearize the uncut pBluescript as an additional size standard??

I really hope this helps, actually I hope that by the time you get this your problems are already solved!! wink.gif

Good LucK!!!


God, I get so tired of people talking about me like I am not here.

Its not my fault I tell you...I'm just made this way.

biggrin.gif biggrin.gif


QUOTE (pBluescript @ Jan 22 2006, 12:08 PM)
God, I get so tired of people talking about me like I am not here.

Its not my fault I tell you...I'm just made this way.

biggrin.gif biggrin.gif

pBluescript, we love you. wub.gif (hope you're knee's on the mend)

in other news, my latest ligation produced absolutely zip. the gel of the ligation (not the mini's, just the ligation mixture) showed lots (about 8-9) bands forming from 3 kb to ~12kb. sad.gif

i'm starting all over again, from the very beginning... Ligand seeks two receptors into binding and mutual phosphorylation. Let's get together and transduce some signals...

For pete's sake, this is a simple ligation, it's not rocket science. smile.gif


Quote: Ligand seeks two receptors into binding and mutual phosphorylation. Let's get together and transduce some signals...